Safety and Immunogenicity of a Newcastle Disease Virus Vector-Based SARS-CoV-2 Vaccine Candidate, AVX/COVID-12-HEXAPRO (Patria), in Pigs

Abstract

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were developed in record time and show excellent efficacy and effectiveness against coronavirus disease 2019 (COVID-19). However, currently approved vaccines cannot meet the global demand. In addition, none of the currently used vaccines is administered intranasally to potentially induce mucosal immunity. Here, we tested the safety and immunogenicity of a second-generation SARS-CoV-2 vaccine that includes a stabilized spike antigen and can be administered intranasally. The vaccine is based on a live Newcastle disease virus vector expressing a SARS-CoV-2 spike protein stabilized in a prefusion conformation with six beneficial proline substitutions (AVX/COVID-12-HEXAPRO; Patria). Immunogenicity testing in the pig model showed that both intranasal and intramuscular application of the vaccine as well as a combination of the two induced strong serum neutralizing antibody responses. Furthermore, substantial reactivity to B.1.1.7, B.1.351, and P.1 spike variants was detected. Finally, no adverse reactions were found in the experimental animals at any dose level or delivery route. These results indicate that the experimental vaccine AVX/COVID-12-HEXAPRO (Patria) is safe and highly immunogenic in the pig model. IMPORTANCE Several highly efficacious vaccines for SARS-CoV-2 have been developed and are used in the population. However, the current production capacity cannot meet the global demand. Therefore, additional vaccines-especially ones that can be produced locally and at low cost-are urgently needed. This work describes preclinical testing of a SARS-CoV-2 vaccine candidate which meets these criteria.

Keywords: COVID-19; HexaPro; NDV; Newcastle disease virus; SARS-CoV-2; coronavirus vaccine; pig model; pigs; prolines; spike; vaccine.

FIG 1

Schematic representation of AVX/COVID-12-HEXAPRO production and testing in pigs. Vector design is shown in panel A. The ectodomain of the spike protein was fused to the transmembrane domain and cytoplasmic tail (TM/CT) of the fusion (F) protein of the NDV by a short GGGGS linker to generate the S/F chimera. The polybasic cleavage site (RRAR) was eliminated by removing the three arginines (R). HEXAPRO stabilizing mutations were introduced into the ectodomain of spike. The construct is designated HXP-S. The nucleotide sequence of HXP-S was codon optimized for mammalian cell expression. The sequence was inserted between the P and M genes in the NDV genome (LaSota strain), which harbors an L289A mutation in the F protein. The experimental design is shown in panel B. Pigs were vaccinated with AVX/COVID-12-HEXAPRO on day 0 and day 21. Blood samples were collected at days 0, 7, 14, 21, 28, and 35 after the first vaccine dose administration. Nasal swabs were collected at days 0, 2, and 22 after the first vaccine dose administration. Distribution of groups is shown in panel C. Ten different groups were included in the study. Classification into IN-IN vaccinated animals (groups 1 to 3), IM-IM vaccinated animals (groups 4 to 7), and other vaccination regimens (groups 8 to 10) is shown. The corresponding dose, volume, and time points of vaccine administration are described. The sample number of pigs per group is indicated.

FIG 2

Kinetics of body temperature in AVX/COVID-12-HEXAPRO-vaccinated pigs. Temperature (°C) of pigs was measured daily 4 days before through 35 days after the vaccine administration. The reference value for fever in pigs (38.8°C) is indicated by the red dotted line. Vaccination time points (prime and boost) are indicated with blue arrows. Mean of daily temperature per group plus standard error of the mean (SEM) is shown.

FIG 3

Antibody responses against SARS-CoV-2 RBD and full-length spike ectodomain in sera from vaccinated pigs. Antibody responses against the receptor binding domain (RBD; panels A, C, E, and G) and the full-length spike protein ectodomain (panels B, D, F, and H) were assessed by ELISA. Responses were measured in the IN-IN groups (A and B), in the IM-IM groups (C and D), and in other vaccination regimens (E and F) at 0, 7, 14, 21, 28, and 35 days after the first vaccination. A comparison of individual points for every pig is shown in panels G and H. In panels G and H, d0-2x refers to the administration of the IN and IM doses simultaneously at day 0, and d0-d21 refers to the IN and IM doses administered at days 0 and 21, respectively. Mean of antibody levels per group expressed as area under the curve (AUC) plus standard error of the mean (SEM) is shown. Ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test was applied to panels G and H. All adjusted P values of <0.05 were considered statistically significant with a confidence interval of 95%. **, P < 0.0011; ***, P < 0.0002; ****, P < 0.0001.

FIG 4

Inhibition of ACE2-RBD interaction by vaccine-induced antibodies. The inhibition of the interaction between the RBD and the receptor angiotensin-converting enzyme 2 (ACE2) was assessed using the ELISA cPass GenScript kit. The 50% inhibitory dilution (ID₅₀) using day 28 (7 days postboost) and day 35 (14 days postboost) samples at a single serum dilution (1:20) is presented (A and B). Samples with titers above the upper level of quantification were serially diluted and retested (C). High-titer COVID-19 convalescent-phase human sera were included as additional positive control. For panels A and B, the limit of detection of the assay (LOD) is indicated with the red dotted line, the positive-control range is indicated with the purple dotted line, and the mean of the ID₅₀ per group plus standard error of the mean (SEM) is shown. In panel C, titers (log₂) are presented.

FIG 5

Neutralization of USA‐WA1/2020 SARS-CoV-2 by antibodies in sera from vaccinated pigs. The neutralization activity of the antibodies contained in sera from vaccinated pigs from all the groups was assessed against the authentic USA‐WA1/2020 SARS-CoV-2 using day 35 postvaccination samples. IN-IN vaccinated animals are shown in brown, IM-IM in green, and other vaccination regimens in blue. Neutralization capacity is expressed as the half-maximum inhibitory dilution (ID₅₀). Mean of the IC₅₀ per group plus standard error of the mean (SEM) is shown. Ordinary one-way ANOVA with Dunnett’s multiple-comparison test was performed. All adjusted P values of <0.05 were considered statistically significant with a confidence interval of 95%. *, P < 0.0138.

FIG 6

Cross-reactivity of vaccine-induced antibodies to the spike protein of variants of concern. The reactivity of vaccine-induced antibodies was assessed by ELISA against the variants of concern P.1, B.1.1.7, and B.1.351 compared to the wild-type (WT) spike protein. IN-IN vaccinated animals are shown in panel A, IM-IM in panel B, and other vaccination regimens in panel C. Mean of antibody levels per group expressed as area under the curve (AUC) is shown.